ProjectSummary Metastasis is the leading cause of cancer-related deaths. But despite much effort in elucidating the molecularmechanismsunderlyingmetastasis,thereisstillmuchthatremainsunknownandunexplored.Inmany solid tumors, metastasis is closely associated with an epithelial-to-mesenchymal transition (EMT), a form of cellular plasticity by which stationary epithelial cells acquire the motile, invasive phenotypes reminiscent of fibroblasts.AndwhileEMTresultsinmanytumorcell-intrinsicchanges,suchchangesarealsoknowntoalter thetumormicroenvironment(TME)inwaysthatfacilitatetumorcellinvasionintothebloodstreamandultimately metastasis. Thus, to define the molecular changes that occur during EMT in vivo, our lab performed whole transcriptomic analysis on sorted epithelial and mesenchymal tumor cells that were harvested from a well- establishedgeneticmousemodelofspontaneouspancreaticductaladenocarcinoma(PDA).Amongthegenes thatweremostsignificantlyupregulatedinmesenchymalcellswasSerping1,whichencodesthehepatocyte- derived plasma protease inhibitor C1-inhibitor (C1-INH). Intriguingly, our preliminary results demonstrate a possible role for tumor-derived C1-INH in the growth and vascularization of mesenchymal PDA tumors. Furthermore,recentstudieshaveshownthatcomplementfactorC1Q,whosedissociationfromthecomplement cascade depends on C1-INH, promotes angiogenesis in the context of wound healing, as well as melanoma progression. Based on our preliminary data and these newer studies, we hypothesize that C1-INH is upregulated in PDA cells during EMT to promote angiogenesis and tumor progression in a C1Q- dependentmanner.Inthisproposal,weprovidedetailsoftwoaimstotestthishypothesis.Briefly,inAim1,we willmoredeeplyinvestigatetheroleoftumor-derivedC1-INHinangiogenesisbyperforminggeneticgainand loss of function studies in murine PDA cells and using standard in vitro and in vivo angiogenesis assays to determine C1-INH?s effects on endothelial cell proliferation, migration, and differentiation, as well as the functional integrity of intratumoral blood vessels. We will also dissect the molecular mechanism of C1-INH in PDAprogressionbyinvestigatingitsfunctionaldependenciesonC1Q.SinceC1-INHwasoriginallyfoundtobe enrichedinthemesenchymalstate,wewillalsoinvestigateitspotentialroleinpromotingmetastasis.Thus,in Aim2,wewilldeterminewhethertumor-derivedC1-INHisnecessaryformetastasisbyemployinganorthotopic transplantationmodelandmonitoringdifferentstagesofthemetastaticcascade,fromtumorcelldissemination to metastatic colonization. The completed aims of this proposal ? which represents the first functional investigationofC1-INHincancer?ultimatelyseektounderstandhowcancercellsinfluencetheTMEtofacilitate tumorgrowthandmetastasis,andmayprovideaspringboardforfuturetherapeuticapplicationanddiscovery.